Process for the production of D-α-amino acids by hydrolysis of the corresponding N-carbamyl derivative

ABSTRACT

In a process for the production of a D-α-amino acid, in which an N-carbamyl-D-α-amino acid corresponding to the general formula: ##STR1## wherein R represents phenyl, hydroxy-substituted phenyl, substituted or unsubstituted alkyl, or thienyl, is converted by a microbial enzyme in an aqueous medium to a D-α-amino acid corresponding to the general formula: ##STR2## wherein R is the same as defined above, decarbamylase produced by a microorganism of the genus Comamonas, Blastobacter, Alcaligenes, Sporosarcina, Rhizobium, Bradyrhizobium or Arthrobacter is used as the enzyme converting the N-carbamyl-D-α-amino acid to the D-α-amino acid. 
     The conversion of the N-carbamyl-D-α-amino acids to the D-α-amino acids is carried out in a neutral to alkaline pH range.

FIELD OF THE INVENTION

The present invention relates to a process for the production of aD-α-amino acid, in which a D-N-carbamyl-α-amino acid corresponding tothe general formula: ##STR3## wherein R represents phenyl,hydroxy-substituted phenyl, substituted or unsubstituted alkylpreferably having 1 to 5 carbon atoms, or thienyl, is converted by anenzyme having an ability to eliminate the carbamyl group (hereinafterreferred to as "decarbamylase") to a D-α-amino acid corresponding to theformula: ##STR4## wherein R is the same as defined above.

These optically active D-α-amino acids are important as pharmaceuticalintermediates. In particular, D-phenylglycine andD-(4-hydroxyphenyl)glycine (hereinafter referred to as "D-HPG") areuseful for the production of semi-synthetic penicillin andsemi-synthetic cephalosporin.

PRIOR ART

Production of D-α-amino acids by eliminating carbamyl groups fromcorresponding D-N-carbamyl-α-amino acids has been known. The eliminationhas been done chemically (Japanese Patent Publication No. 4707/1983) orby enzymatic reactions of microorganisms (Japanese Patent PublicationNos. 18793/1982, 20520/1988 and 48758/1989).

Most of these microbial enzymes have optimum pH values around a neutralrange. However, in the previous step, wherein the N-carbamyl-D-α-aminoacids are prepared by selectively hydrolyzing the D-form ofcorresponding DL-5-substituted hydantoins, hydantoin hydrolase havingoptimum pH of from 8 to 9 is used. Therefore, the subsequent conversionof the N-carbamyl-D-α-amino acids to D-α-amino acids by decarbamylasehas to be carried out in a different reaction medium from that in theprevious step.

SUMMARY OF THE INVENTION

The present inventors made a search for microorganisms which couldenzymatically eliminate carbamyl groups ("decarbamylation") fromD-N-carbamyl-α-amino acids to form D-α-amino acids, mainly among thegenera which are unknown to have decarbamylase. It has now been foundthat the bacteria of the genera Comamonas, Blastobacter, Alcaligenes,Sporosarcina, Rhizobium, Bradyrhizobium and Arthrobacter have thedecarbamylase activity.

Accordingly, the present invention relates to a process for theproduction of a D-α-amino acid, in which an N-carbamyl-D-α-amino acidcorresponding to the general formula (I): ##STR5## wherein R representsphenyl, hydroxy-substituted phenyl, substituted or unsubstituted alkylpreferably having 1 to 5 carbon atoms, or thienyl, is converted by amicrobial enzyme in an aqueous medium to a D-α-amino acid correspondingto the general formula (II): ##STR6## wherein R is the same as definedabove, characterized in that the enzyme is decarbamylase produced by amicroorganism of the genus Comamonas, Blastobacter, Alcaligenes,Sporosarcina, Rhizobium, Bradyrhizobium or Arthrobacter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the results of decarbamylation at 50° C. and30° C. by newly screened strains.

FIG. 2 is a graph showing the effects of various additives to theculture medium of Comamonas sp. E 222 C on its decarbamylaseproductivity.

FIG. 3 is a result of SDS-polyacrylamide gel electrophoresis of purifiedComamonas sp. E 222 C decarbamylase.

FIG. 4 is a result of gel filtration of the purified Comamonas sp. E 222C decarbamylase thorough a Sephadex G-150 column.

FIG. 5 is a graph showing the effect of pH on the activity of thepurified Comamonas sp. E 222 C decarbamylase.

FIG. 6 is a graph showing the effect of temperature on the activity ofthe purified Comamonas sp. E 222 C decarbamylase.

FIG. 7 is a graph showing the effects of various additives to theculture medium of Blastobacter sp. A 17 p-4 on its decarbamylaseproductivity.

FIG. 8 is a graph showing the effects of various metal ions added to theculture medium of Blastobacter sp. A 17 p-4 on its decarbamylaseproductivity.

FIG. 9 is a result of SDS-polyacrylamide gel electrophoresis of purifiedBlastobacter sp. A 17 p-4 decarbamylase.

FIG. 10 is a result of gel filtration of the purified Blastobacter sp. A17 p-4 decarbamylase thorough a Sephadex G-150 column.

FIG. 11 is a graph showing the effect of pH on the activity of thepurified Blastobacter sp. A 17 p-4 decarbamylase.

FIG. 12 is a graph showing the effect of temperature on the activity ofthe purified Blastobacter sp. A 17 p-4 decarbamylase.

DETAILED EXPLANATION OF THE INVENTION

According to the present invention, both D-isomer specific decarbamylaseand unspecific decarbamylase may be actually used. An enzyme which isstrictly stereoselective to D-N-carbamyl-α-amino acids is often calledD-N-carbamyl-α-amino acid amide hydrolase.

Among the microorganisms according to the present invention, strainsshowing particularly high decarbamylase activity are, for example,Comamonas sp. E 222 C (FERM BP No. 4411, National Institute ofBioscience and Human-Technology, Agency of Industrial Science andTechnology, 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, 305, Japan,Sep. 18, 1992), Comamonas sp. E 206 a, Comamonas sp. E 217 a,Blastobacter sp. NA 88-b, Blastobacter sp. A 17 p-4 (FERM BP No. 4410,National Institute of Bioscience and Human-Technology, Agency ofIndustrial Science and Technology, Sep. 18, 1992), Alcaligenes sp. E 215(FERM BP No. 4409, National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology, Sep. 18,1992), Alcaligenes xylosoxidans subsp. denitrificans CL 66-2 a,Sporosarcina sp. NCA 28-b (FERM BP No. 4408, National Institute ofBioscience and Human-Technology, Agency of Industrial Science andTechnology, Sep. 24, 1992), Rhizobium sp. KNK 1415 (FERM BP No. 4419,National Institute of Bioscience and Human-Technology, Agency ofIndustrial Science and Technology, Sep. 22, 1993), Bradyrhizobiumjaponicum IFO 14783, Bradyrhizobium sp. IFO 15003, Arthrobacter sp. CA17-2 (FERM BP No. 4420, National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology, Sep. 22,1993).

Typical examples of the strains Comamonas sp. E 222 C, Blastobacter sp.A 17 p-4, Alcaligenes sp. E 215, Sporosarcina sp. NCA 28-b, Rhizobiumsp. KNK 1415 and Arthrobacter sp. CA 17-2 are microbiologicallycharacterized as follows:

    ______________________________________    Comamonas sp. E 222 C    ______________________________________    (a) Morphology    Bacillus (bent), Gram-variable    Spore: -    Colony:          buff, semi-translucent, round, regular, entire,          shiny, low convex, smooth; diameter 1 mm (48 hr)    Growth (48 hr) at:              37° C. +              41° C. -              45° C. -    (b) Physiological activities    Catalase             +    Oxidase              -    Glucose fermentation -    NO.sub.3 Reduction   -    Indole production    -    Acid production from glucose                         -    Arginine dehydrolase -    Urease               -    Aesculin hydrolysis  -    Gelatin hydrolysis   -    βGalactosidase  -    Cytochrome oxidase   + (weak)    (c) Assimilation    Glucose              -    Arabinose            -    Mannose              -    Mannitol             -    N-Acetylglucosamine  -    Maltose              -    Gluconic acid        +    Caproic acid         +    Adipic acid          -    Maltic acid          +    Citric acid          +    Phenyl acetate       +    ______________________________________

    ______________________________________    Blastobacter sp. A 17 p-4    ______________________________________    (a) Morphology    Bacillus (oval), Gram-negative    Spore: -    Colony:          white, semi-translucent, round, regular, entire,          shiny, low convex, smooth, mucoid producing;          diameter 1.0-1.5 mm (48 hr)    Growth (48 hr) at:              37° C. +              41° C. -              45° C. -    (b) Physiological activities    Catalase             +    Oxidase              + (weak)    Glucose fermentation -    NO.sub.3 Reduction   -    Indole production    -    Acid production from glucose                         -    Arginine dehydrolase -    Urease               +    Aesculin hydrolysis  +    Gelatin hydrolysis   -    β-Galactosidase +    Cytochrome oxidase   +    (c) Assimilation    Glucose              +    Arabinose            +    Mannose              +    Mannitol             +    N-Acetylglucosamine  +    Maltose              +    Gluconic acid        -    Caproic acid         -    Adipic acid          -    Maltic acid          -    Citric acid          -    Phenyl acetate       -    ______________________________________

    ______________________________________    Alcaligenes sp. E 21    ______________________________________    (a) Morphology    Bacillus, Gram-variable    Spore: -    Mobility: +    Colony:          buff, semi-translucent, round, regular, entire,          low convex, shiny, smooth; diameter 1 mm (48 hr)    Growth (48 hr) at:              37° C. +              41° C. -              45° C. -    (b) Physiological activities    Catalase             +    Oxidase              -    Glucose fermentation -    ______________________________________

    ______________________________________    Sporosarcina sp. NCA 28-b    ______________________________________    (a) Morphology    Coccus, Gram-variable    Spore: +    Mobility: -    Colony:          round, regular, entire, cream to white, semi-          translucent; diameter <0.5 mm (48 hr)    Growth at:           37° C. -           45° C. -    (b) Physiological activities    Catalase             +    Oxidase              +    Glucose fermentation -    ______________________________________

    ______________________________________    Rhizobium sp. KNK-1415 (FERM BP-4419)    ______________________________________    (a) Morphology    Bacillus (short), Gram-negative    Spore: -    Flagellum: polar or peritrichous    Colony:          off-white, opaque, entire, regular, smooth, low          convex, mucoid producing; diameter 5 mm (5 days)    Growth at:           30° C.  +           37° C. (±)    (b) Physiological activities    Catalase             +    Oxidase              +    Glucose fermentation -    NO.sub.3 Reduction   +    Indole production    -    Acid production from glucose    Arginine dehydrolase -    Urease               +    Aesculin hydrolysis  +    Gelatin hydrolysis   -    β-Galactosidase +    Cytochrome oxidase   +    3-Ketolactose production                         -    H.sub.2 S Production -    (c) Assimilation    Glucose              +    Arabinose            +    mannose              +    Mannitol             +    N-Acetylglucosamine  +    Maltose              +    Gluconic acid        -    Caproic acid         -    Adipic acid          -    Maltic acid          +    Citric acid          -    Phenyl acetate       -    GC content: 63.4%    Quinone analysis:              Q-10   95.7%              Q-9     3.3%              Q-11    0.6%              Q-8     0.4%    Microbial fatty acid analysis:    (whole fatty acids)           Component                   Area (%)           3-OH C14:0                   4.6           C16:1   5.9           C16:0   14.0           3-OH C16:0                   0.1           C18:1   70.7           C18:0   3.3           unknown 1.4    (3-OH fatty acids)           Component                   Area (%)           3-OH C14:0                   88.8           3-OH C16:0                   6.6           3-OH C18:0                   4.6    (2-OH fatty acid)           not detected    ______________________________________

    ______________________________________    Arthrobacter sp. CA 17-2 (FERM BP-4420)    ______________________________________    (a) Morphology    Bacillus or coccus, Gram-positive    Spore: -    Mobility: +    Colony:          off-white, opaque, round, regular, convex, smooth;          diameter ca. 0.5 mm (2 days)    Growth at:           30° C.  +           37° C. (+)           45° C.  -    (b) Physiological activities    Catalase             +    Oxidase              -    Glucose fermentation -    (c) Chemotaxonamy    Amino acid in peptidoglycan: L-Lysine    Mycolic acid: -    Fatty acid composition:    anteiso C.sub.15:0 (12-methyltetradecanoic acid)                              58%    iso C.sub.15:0 (13-methyltetradecanoic acid)                             5.5%    iso C.sub.16:0 (14-methylpentadecanoic acid)                               8%    anteiso C.sub.17:0 (14-methylhexadecanoic acid)                              25%    ______________________________________

The enzyme decarbamylase can be obtained by cultivating themicroorganisms usually in a liquid medium in a conventional manner.However, a solid medium may also be used. The medium usually containsassimilable carbon and nitrogen sources as well as inorganic saltsessential for the growth of each microorganism. Decarbamylaseproductivity of the bacteria can be preferably improved by adding to theculture medium a small amount of substance such as an amino acid, forexample D-p-hydroxyphenylglycine or D-phenylglycine; anN-carbamyl-α-amino acid, for example N-carbamyl-DL-methionine orN-carbamyl-D-phenylalanine; 5-substituted hydantoin, for exampleDL-5-p-hydroxyphenylhydantoin or DL-5-phenylhydantoin; a pyrimidinemetabolite, for example uracil, dihydrouracil or β-ureidopropionic acid;a metal ion, for example Fe²⁺, Fe³⁺, Be²⁺, Co²⁺, Al³⁺, Li⁺, Mn²⁺, Mg²⁺or Cs⁺, or urea. These additives can be present in the culture medium ina concentration of from 0.1 to 10 mM for the metal ions, and from 0.01to 1% by weight for the other substrates.

The cultivation can be performed at a temperature in the range of from20 to 85° C. and at a pH value in the range of from 4 to 11. Aerationand stirring may promote the growth of microorganisms.

After the cultivation, the culture medium or the microorganismscollected therefrom, as such or further processed, may be served as anenzyme source for decarbamylation of the D-N-carbamyl-α-amino acids.Live or dried (for example, lyophilized) bacteria can be used. They mayalso be served after homogenization, extraction, treatment with asurfactant, or sonication. In addition, pure or crude enzyme recoveredfrom the bacteria homogenate, extract etc. can be used. The pure orcrude enzyme can be immobilized according to, for example, thedisclosure of the International Application PCT/JP91/01696.

In principle, enzymes produced by gene-manipulated microorganisms areequally useful as those from the original bacteria.

The decarbamylation reaction is performed in an aqueous medium at a pHvalue in the range of from 6 to 11, preferably from 7 to 9.5. Someenzymes have optimum pH values of from 8 to 9. Decarbamylation takesplace usually at a temperature of from 20 to 85° C., the temperaturebeing determined optimally for the particular enzyme.

In the present process, the substrates for decarbamylase areN-carbamyl-D-α-amino acids such as D-N-carbamylalanine,D-N-carbamylmethionine, D-N-carbamylvaline, D-N-carbamylleucine,D-N-carbamylphenylglycine, D-N-carbamyl-(4-hydroxyphenyl)glycine,D-N-carbamyl-(2-thienyl)-glycine and D-N-carbamylphenylalanine. Thegroup R in the above mentioned formulae can be, for example, phenyl-,indolyl-, alkylthio-, hydroxyl-, amino- or carboxyl-substituted alkyl.

D-α-Amino acids, the decarbamylation products, can be recovered byconventional procedures such as concentration, neutralization andion-exchange chromatography. In order to separate a relativelyhydrophobic D-α-amino acid such as D-phenylglycine,D-(4-hydroxyphenyl)glycine, D-leucine or D-phenylalanine, thedecarbamylation reaction mixture may be acidified or alkalized toprecipitate impurities off, and then treated in a conventional manner,for example by concentration and neutralization, to precipitate theamino acids. Separation of a relatively hydrophilic products such asD-thienylglycine, D-serine and D-alanine can be achieved by ion-exchangechromatography. The eluate, for example with an ammonia solution, isthen neutralized and concentrated.

The N-carbamyl-D-amino acids as the substrates for decarbamylase can bederived from 5-substituted hydantoin by an enzymatic reaction. As theenzyme source for this reaction, culture media of variousmicroorganisms, the microorganisms collected therefrom (as such orfurther processed), or enzymes extracted therefrom may be used. Theseprocesses are disclosed, for example, in Japanese Patent KokaiPublication Nos. 44690/1978, 69884/1970, 91189/1978, 133688/1978,84086/1979 and 7001/1980.

The D-carbamyl-α-amino acids are converted to corresponding opticallyactive D-α-amino acids by the present process.

EXAMPLES

The present invention is illustrated by the following Examples.

Example 1

Soli samples (taken from different places in Japan) were placed into 2ml of a medium A (1 g/l KH₂ PO₄, 1 g/l K₂ HPO₄, 0.3 g/l MgSO₄ ·7H₂ O,0.1 g/l yeast extract, 1 g/l NH₄ Cl, 1.5 g/l a carbon source as listedbelow: pH 7.0) or a medium B (1 g/l KH₂ PO₄, 1 g/l K₂ HPO₄, 0.3 g/lMgSO₄ ·7H₂ O, 0.5 g/l glucose, 0.1 g/l yeast extract, 1.5 g/l a nitrogensource as listed below; pH 7.0) and aerobically incubated at 28° C.Microorganisms were multiplicated through subculture in the same medium.Then, the culture medium was spread on an agar plate (containing 2% agarin medium A or B) and incubated at 28° C. for 2 to 6 days to isolate thestrains.

Carbon and Nitrogen Sources

N-Carbamyl-D-p-hydroxyphenylglycine (C-D-HPG)

N-Carbamyl-D-phenylglycine (C-D-PG)

N-Carbamyl-D-alanine (C-D-Ala)

DL-5-Methylhydantoin (DL-Ala-hyd)

DL-5-Phenylhydantoin (DL-PG-hyd)

DL-5-p-Hydroxyphenylhydantoin (DL-HPG-hyd)

Citrulline

Phenylurea

n-Butyl carbamate

The isolated strain was suspended in 100 μl of a substrate solution (35mM C-D-HPG, 200 mM potassium phosphate; pH 7.0) and incubated for 1 to 3days at 28° C. Then, a sample of the suspension was analyzed for thecarbamyl-D-HPG and D-HPG contents by TLC on a Merck 60 F₂₅₄ plate. Thesample was developed by butanol:acetic acid:water 3:3:1 (v/v/v). Theamino acids were detected with a UV lump (254 nm) or by sprayingp-(dimethylamino)-cinnamaldehyde and ninhydrin. It was found that 1868strains could assimilate the carbon or nitrogen source as used. Amongthem, 37 strains produced D-HPG, 16 strains of which brought high D-HPGyields (≧2 mM).

Example 2

The 16 decarbamylase-producing strains were subjected to a furtherscreening. For this purpose, they were cultured in 1 ml of a medium C(1.5 g/l C-D-HPG, 1 g/l KH₂ PO₄, 1 g/l K₂ HPO₄, 0.3 g/l MgSO₄ ·7H₂ O, 3g/l yeast extract, 3 g/l meat extract, 10 g/l glycerol, 2 g/lpolypeptone; ph 7.0) for 3 days at 28° C. Then, 1 ml of the culturemedium was centrifuged to collect the microorganism, which was suspendedin 100 μl of the same substrate solution as used in Example. After 24hours at 30° C. or 50° C., the suspension was analyzed for the D-HPGcontent.

A sample of the suspension was subjected to HPLC with a Cosmosil 5 C 18column (4.6×250 mm, Nacarai Tesque). D-HPG was eluted withwater:acetonitrile:phosphoric acid 95:5:0.01 (v/v/v). The D-HPGconcentration was determined from absorbance at 254 nm. The results areshown in FIG. 1.

Among the 16 strains, the three strains E 206 a, E 222 C and E 215,which were grown on C-D-Ala as a single carbon source, were very activeat 30° C. The D-HPG yield by E 222 C was 30.8 mM at 30° C., while only3.5 mM at 50° C. Other three strains A 17 p-1, A 17 p-3 and A 17 p-4,which were cultured on C-D-HPG as a single carbon source, were highlyactive at 50° C. The D-HPG yield by A 17 p-4 amounted to 28.8 mM at 50°C., while only 2.5 mM at 30° C.

Example 3

Substrate Specificity of Decarbamylase

For the determination of substrate specificity of decarbamylase ofComamonas sp. E 222 C and Blastobacter sp. A 17 p-4, these strains werecultured at 28° C. in the medium C (as described in Example 2) for 1 day(E 222 C) and 7 days (A 17 p-4). Three ml of the culture medium wascentrifuged to separate the microorganism, which was suspended in 300 μlof a substrate solution (200 mM potassium phosphate, 1% (w/v) asubstrate as listed below) and incubated for 24 hours at 30° C. (E 222C) or 50° C. (A 17 p-4).

As shown in Table 1, both microorganisms had high activity to hydrolyzethe N-carbamyl substituted, aliphatic and aromatic D-amino acids.However, N-carbamyl derivatives of the amino acids having polar groupswere less hydrolized. Comamonas sp. E 222 C could hydrolizeβ-ureidopropionic acid. It was also found that Blastobacter sp. A 17 p-4had a hydantoinase activity.

                  TABLE 1    ______________________________________    Substrate specificity of novel decarbamylase                   Substrate conversion                     Comamonas Blastobacter    Substrate        sp. E 222 c                               sp. A 17 p-4    ______________________________________    N-Carbamyl-    D-alanine        ++++      +++    D-valine         ++++      +++    D-leucine        ++++      +++    D-serine         +         -    D-phenylalanine  ++++      +++    D-phenylglycine  +++       +++    D-p-hydroxyphenylglycine                     +++       ++    DL-norvaline     ++        ++    DL-norleucine    ++        ++    DL-methionine    ++        ++    DL-threonine     +         -    sarcosine        -         -    β-Ureidopropionic acid                     ++++      -    Ureidosuccinic acid                     -         -    DL-5-Methylhydantoin                     -         ++    DL-5-Phenylhydantoin                     -         ++    DL-5-p-Hydroxyphenyl-                     -         ++    hydantoin    ______________________________________     Conversion to amino acids:     -: 0%     +: <10%     ++: <50%     +++: <80%     ++++: 100%

Example 4

Promoters for the Production of Comamonas sp. E 222 C Decarbamylase

A search was made for the substances which could increase the yield ofdecarbamylase of Comamonas sp. E 222 C.

The strain was cultured in a nutrient medium D (1 g/l KH₂ PO₄, 1 g/l K₂HPO₄, 0.3 g/l MgSO₄ ·7H₂ O, 3 g/l yeast extract, 3 g/l meat extract, 10g/l glycerol, 2 g/l polypeptone; pH 7.0) for 2 days at 28° C. Theculture medium additionally contained a substance as listed in FIG. 2 ina concentration of 0.15% (w/v).

One ml of the culture medium was centrifuged to collect themicroorganism, which was suspended in 100 μl of a substrate solution (35mM C-D-HPG, 200 mM potassium phosphate; pH 7.0). After 24 hours at 30°C., D-HPG was quantitated by HPLC according to the procedure describedin Example 2. It was found, as shown in FIG. 2, that the decarbamylaseproductivity could be improved by urea, β-ureidopropionic acid,D-phenylglycine or N-carbamyl-DL-methionine.

Example 5

Purification of Comamonas sp. E 222 C Decarbamylase

Comamonas sp. E 222 C was cultured at 28° C. for 3 days in the medium D(as defined in Example 4) containing 0.15% (w/v) of β-ureidopropionicacid. Total 3.6 l of the culture medium was centrifuged to collect themicroorganism, which was suspended in 100 ml of 10 mM potassiumphosphate (pH 7.0), homogenized by sonication and then centrifuged totake the supernatant containing the enzyme. Ammonium sulfate was addedto the enzyme solution. Precipitation fraction at an ammonium sulfateconcentration of 20-40% saturation was centrifuged to separate theenzyme, which was dissolved in the same buffer solution as used above.

Then, 177 ml of the enzyme solution was dialyzed to 10 l of the samebuffer for 12 hours, charged into a DEAE-Sephacel column (5×15 cm), andeluted with linear gradient 0 to 1 M NaCl. The eluate was adjusted to anNaCl concentration of 4M, charged into a Phenyl-Sepharose CL-4B column(1.5×15 cm), and eluted with linear gradient 4 to 0 M NaCl. Thusobtained active fraction was concentrated by ultrafiltration through aYM-10 membrane (Amicon).

The concentrate (5 ml) was gel-filtrated through a Sephacryl S-200 HRcolumn (1.8×80 cm) with the same buffer solution as used aboveadditionally containing 0.2 M NaCl. The active fraction was desalted bydialysis, charged into a hydroxyapatite column (1.2×10 cm), eluted withlinear gradient 0 to 1 M potassium phosphate (pH 7.0). The active eluate(8.5 ml) was dialyzed for 12 hours to 500 ml of 10 mM potassiumphosphate (pH 7.0), charged into a Mono Q HR 5/5 column, and eluted withlinear gradient 0 to 1.0 M NaCl. The thus obtained active fraction (1.2ml) was analyzed as a purified enzyme solution.

As shown in Table 2, specific activity of the purified enzyme solutionwas 108 times that of the supernatant of homogenized cell suspension.Enzyme activity recovery was about 2%. Ten mg of the purified enzyme wassubjected to SDS-polyacrylamide gel electrophoresis (10% polyacrylamide)according to the King and Laemmli's method (King, J., Laemmli, U. K.,Journal of Molecular Biology, Vol. 62, 165-477 (1971)). A single bandwas detected at around MW 38,000, as shown in FIG. 3.

                  TABLE 2    ______________________________________    Purification of Comamonas sp. E 222 c decarbamylase                          Enzyme   Specific                  Protein activity activity                                          Recovery    Purification step                  (mg)    (U)      (U/mg) (%)    ______________________________________    1)  Supernatant of                      6316    23.5   0.0037 100        cell homogenate    2)  Ammonium sulfate                      2362    21.2   0.0090 90.2        precipitation        fraction    3)  DEAE-Sephacel 146     21.6   0.15   91.9        fraction    4)  Phenyl-Sepharose                      40.0    10.9   0.27   46.4        fraction    5)  Sephacryl S-200                      23.8    6.6    0.28   29.1        HR fraction    6)  Hydroxyapatite                      2.5     0.7    0.28   3.0        fraction    7)  Mono Q HR 5/5 1.2     0.48   0.40   2.0        fraction    ______________________________________

In addition, the purified enzyme was subjected to gel filtration througha Sephadex G-150 column (1.5×85 cm with 10 mM potassium phosphate (pH7.0) containing 0.2 M NaCl and 0.1 mM dithiothreitol (DTT). The enzymewas eluted at around MW 111,000, as shown in FIG. 4.

Example 6

Characteristics of Comamonas sp. E 222 C Decarbamylase

Optimum pH and temperature of Comamonas sp. E 222 C decarbamylase weredetermined by using the enzyme solution obtained in Example 5.

Optimum pH was determined as follows: The enzyme solution was contactedwith 10 mM N-carbamyl-D-phenylalanine which was buffered with 200 mMacetate-hydrochloride (pH 4.2-5.9), potassium phosphate (pH 5.0-8.8),Tris-hydrochloride (pH 7.6-9.9) or glycine-NaOH (pH 8.9-11.1). (Thevolume of the mixture was 500 μl.) After the reaction time of 20 minutesat 30° C., the reaction was terminated by the addition of 500 μl ofethanol.

To quantitate D-phenylalanine formed, a sample of the reaction mixturewas added to a solution containing 200 mM potassium phosphate (pH 7.0),1.5 mM 4-aminoantipyrine, 2.1 mM phenol, 2.25 u of peroxidase (obtainedfrom horseradish, CALZYME Lab.) and 0.375 units of D-amino acid oxidase(Sigma) to make the total volume 500 μl. After the mixture was incubatedat 37° C. for 60 minutes, increase of absorbance at 500 nm was measured.

Optimum temperature was determined as follows: The enzyme solution wascontacted with the same substrate solution as used above (buffered withpotassium phosphate; pH 7.0) for 20 minutes at 10-80° C. Then, ammoniaformed was quantitated by the indophenol method (Bollter, W. T. et al.,Analytical Chemistry, Vol. 33, 592-594 (1961)).

The results are shown in FIG. 5 and FIG. 6. It was found that theoptimum pH and temperature of Comamonas sp. E 222 C decarbamylase were8-9 and 40° C.

Example 7

Amino Acid Sequence of Comamonas sp. E 222 C Decarbamylase

Amino acid sequence of the protein decarbamylase was determined by usingthe enzyme solution obtained in Example 5. The enzyme solution wasdesalted with Centricon-10 Microcentrater (Amicon) and charged into apulse-liquid protein sequencer. The enzyme had the following amino acidsequence at amino terminal (Arg at 20 and 26 were not confirmed due tounclear peaks)

Example 8

Promoters for the Production of Blastobacter sp. A 17 p-4 Decarbamylase

A search was made for the substances which could increase the yield ofdecarbamylase of Blastobacter sp. A 17 p-4.

The strain was cultured at 28° C. for 3 days in the medium D (as definedin Example 4) additionally containing 0.15% (w/v) of a substance aslisted in FIG. 7. Then, D-HPG was formed in the same way as in Example 4but that the reaction temperature was 40° C., and quantitated by HPLC.As shown in FIG. 7, the decarbamylase yield was increased in thepresence of uracil, dihydrouracil or β-ureidopropionic acid.

In order to examine the effects of metal ions on the enzymeproductivity, the strain was cultured at 28° C. for 4 days in the mediumD additionally containing 0.15% (w/v) of uracil and 2 mM a metal ion aslisted in FIG. 8. It was found by quantitating D-HPG that ions such asFe²⁺, Fe³⁺, Li⁺, Cs⁺, Be²⁺, Mg²⁺, Mn²⁺, Co²⁺ and Al³⁺ increased thedecarbamylase yield.

Example 9

Purification of Blastobacter sp. A 17 p-4 Decarbamylase

Blastobacter sp. A 17 p-4 was cultured at 28° C. for 7 days in themedium D (as defined in Example 4) which additionally containing 0.15%(w/v) of uracil and 2 mM FeSO₄ ·7H₂ O. Total 4.8 l of the culture mediumwas centrifuged to separate the microorganism, which was suspended in120 ml of 10 mM potassium phosphate, homogenized at 5° C. for 20 minuteswith glass beads (diameter: 0.25 mm; Dyno-Mill KDL, Switzerland) andcentrifuged to obtain a crude enzyme solution as a supernatant. Ammoniumsulfate was added to the enzyme solution to take a precipitationfraction at an ammonium sulfate concentration of 20-40% saturation. Theprecipitate was separated by centrifugation and dissolved in the samebuffer solution as used above.

Then, 41 ml of the enzyme solution was dialyzed for 12 hours to 5 l ofthe same buffer solution as used above, purified with a DEAE-Sephacelcolumn and a Phenyl-Sepharose CL-6B column, and concentrated byultrafiltration in the same way as in Example 5. The concentrate (3 ml)was gel-filtrated through a Sephadex G-150 column (1.5×80 cm) with thesame buffer solution as used above (additionally containing 0.2 M NaCl),desalted by dialysis, and purified by the same procedure as in Example 5with a Mono Q HR 5/5 column. The thus obtained active fraction (2 ml)was analyzed as a purified enzyme solution. As shown in Table 3,specific activity of the purified enzyme solution was 37 times that ofthe supernatant of homogenized cell suspension. Enzyme activity recoverywas 2.3%.

                  TABLE 3    ______________________________________    Purification of Blastobacter sp. A 17 p-4 decarbamylase                          Enzyme   Specific                  Protein activity activity                                          Recovery    Purification step                  (mg)    (U)      (U/mg) (%)    ______________________________________    1)  Supernatant of                      4657    52.0   0.011  100        cell homogenate    2)  Ammonium sulfate                      797     30.2   0.038  58.1        precipitation        fraction    3)  DEAE-Sephacel 284     18.3   0.064  35.2        fraction    4)  Phenyl-Cellulose                      89.6    7.6    0.085  14.6        fraction    5)  Sephadex G-150                      15.4    4.1    0.27   7.9        fraction    6)  Mono Q HR 5/5 2.9     1.2    0.41   2.3        fraction    ______________________________________

The purified enzyme was subjected to SDS-polyacrylamide gelelectrophoresis. A single band was detected at around MW 39,000; seeFIG. 9. In addition, the purified enzyme was gel-filtrated through aSephadex G-150 column in the same way as in Example 5. The enzyme waseluted at around MW 120,000; see FIG. 10.

Example 10

Characteristics of Blastobacter sp. A 17 p-4 Decarbamylase

Optimum pH and temperature of Blastobacter sp. A 17 p-4 decarbamylasewere determined by using the enzyme solution obtained in Example 9according to the same procedure as in Example 6. The results are shownin FIGS. 11 and 12. It was found that the optimum pH and temperature ofBlastobacter sp. A 17 p-4 decarbamylase were 9 and 50° C.

Example 11

Amino Acid Sequence of Blastobacter sp. A 17 p-4 Decarbamylase

Amino acid sequence of the protein decarbamylase was determined by usingthe enzyme solution obtained in Example 9 according to the sameprocedure as in Example 7. The enzyme had the following amino acidsequence at amino terminal (Arg at 38 was not confirmed due to unclearpeak) (Seq. ID No: 2):1 5 1015Ala-Arg-Lys-Leu-Asn-Leu-Ala-Val-Ala-Gln-Leu-Gly-Pro-Ile-Ala- 20 2530Arg-Ala-Glu-Thr-Arg-Asp-Gln-Val-Val-Ala-Arg-Leu-Met-Glu-Met- 3540Met-Lys-Glu-Ala-Lys-Ser-Ser-(Arg)-Gly-Thr

Example 12

The seven strains of the genera Rhizobium and Bradyrhizobium listed inTable 4 were cultured at 30° C. for 24 hours in 1 ml of the 805 liquidmedium (yeast extract 1 g/l, mannitol 5 g/l, K₂ HPO₄ 0.7 g/l, KH₂ PO₄0.1 g/l, MgSO₄ ·7H₂ O 1 g/l, C-D-HPG 1 g/l; pH 7.0) containing C-D-HPGor C-D-Ala in a final concentration of 1 g/l. The culture medium wascentrifugated to separate the microorganism, which was suspended in 0.5ml of a substrate solution (1% C-D-HPG or C-D-Ala, 0.1 M potassiumphosphate (pH 7.0), 0.1% Triton X-100). The suspension was incubated at37° C. for 24 hours and analyzed by TLC in the same way as in Example 1.Decarbamylation activity was found in the strains of genusBradyrhizobium, as shown in Table 4.

                  TABLE 4    ______________________________________    Strain              Decarbamylase activity    ______________________________________    Rhisobium lezi IFO 14779                        ++    Rhisobium meliloti IFO 14782                        ++    Rhisobium Fredii IFO 14780                        ++    Rhisobium galegae IFO 14965                        ++    Rhisobium huakuii IFO 15243                        +    Bradyrhyzobium japonicum IFO 14783                        +    Bradyrhyzobium sp. IFO 15003                        +    ______________________________________

Example 13

Soil samples were subjected to another screening in the same way as inExample 1. N-Carbamyl-D-leucine (C-D-Leu), N-carbamyl-D-alanine(C-D-Ala), N-carbamyl-D-phenyl-glycine (C-D-PG) or DL-5-methylhydantoin(DL-Ala-hyd) was used as a carbon or nitrogen source. Among the strainswhich were grown, 9 strains were tested for decarbamylase activity onC-D-Ala or C-D-HPG according to the procedure of Example 12. The resultsare shown in Table 5. Some strains were more specific to C-D-HPG than toC-D-Ala, and the others more specific to C-D-Ala than to C-D HPG.

                  TABLE 5    ______________________________________    Strain           Substrate  Activity    ______________________________________    Rhizobium sp. KNK 1415                     C-D-PG     +++ (C-D-HPG)    Alcaligenes xylosoxidans    subsp. dentrificans    CL 66-2a         C-D-Leu    ++ (C-D-HPG)    CL 67-1          C-D-Leu    ++ (C-D-HPG)    CL 85-1          C-D-Leu    + (C-D-HPG)    CA 17-1          C-D-Ala    + (C-D-Ala)    Arthrobacter sp.    CA 17-2          C-D-Ala    ++ (C-D-Ala)    CA 77-2          C-D-Ala    ++ (C-D-Ala)    AH 71-1          DL-Ala-hyd + (C-D-Ala)    AH 57-1          DL-Ala-hyd ++ (C-D-Ala)    ______________________________________

Example 14

The strain of Rhyzobium sp. KNK 1415 which was obtained in Example 13was cultured at 30° C. for 40 hours in 10 l of the SE medium (sucrose 23g/l, yeast extract 4 g/l, urea 2 g/l, KH₂ PO₄ 2 g/l, Na₂ HPO₄ 2 g/l,MgSO₄ ·7H₂ O 1 g/l, MnCl₂ ·4H₂ O 0.01 g/l; pH 6.5). The culture mediumwas centrifugated to separate the microorganism, which was washed with0.9% saline, homogenized by sonication, and centrifugated. Thesupernatant was treated with protamine sulfate (0.1 mg/mg protein) forenucleation and then centrifugated. The supernatant was heated to 50° C.for 30 minutes and centrifugated to remove denatured proteins. To thesolution, ammonium sulfate was added in a concentration of 30%saturation. The ammonium sulfate precipitate was collected bycentrifugation, dissolved in 500 ml of a buffer solution (20 mM Tris·HCl(pH 7.5), 2 mM DTT), dialyzed to the same buffer solution, charged intoa DEAE-cellulose column and eluted with a solution (10 mM sodiumphosphate (pH 7.2), 0.15 M NaCl, 1 mM DTT). The eluate was concentratedby ultrafiltration with a YM-10 membrane (Amicon) and analyzed bySDS-polyacrylamide gel electrophoresis. The enzyme decarbamylase wasdetected at around 35,000.

Example 15

The part of decarbamylase band of the SDS-polyacrylamide gel fromExample 14 was cut out, homogenized in a buffer solution (50 mM Tris·HCl(pH 7.5), 0.1% SDS, 0.1 mM EDTA, 150 mM NaCl, 5 mM DTT) and eluted atroom temperature. The extract was concentrated by ultrafiltration,charged into a reverse phase HPLC column (AP-303; YMC) and eluted withgradient acetonitril. Thus obtained decarbamylase-containing fractionwas charged into a gas phase protein sequencer (Applied Biosystems). Theenzyme had the following amino acid sequence at amino terminal (Seq. IDNo: 3):1 5 10Thr-Arg-Gln-Met-Ile-Leu-Ala-Val-Gly-Gln-

Effects of the Invention

By using novel decarbamylase according to the invention, D-α-aminoacids, which are important intermediates for the production ofpharmaceuticals such as antibiotics, can be prepared more efficientlyunder convenient conditions such as pH 8-9 and 50° C.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 3    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 30 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    -      Ser Arg Ile Val Asn Tyr Ala Ala - # Ala Gln Leu Gly Pro Ile Gln    Arg    #   15    -      Ala Asp Ser Arg Ala Asp Val Met - # Glu Arg Leu Leu Ala His    #                 30    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 40 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    -      Ala Arg Lys Leu Asn Leu Ala Val - # Ala Gln Leu Gly Pro Ile Ala    Arg    #   15    -      Ala Glu Thr Arg Asp Gln Val Val - # Ala Arg Leu Met Glu Met Met    Lys    #                 30    -      Glu Ala Lys Ser Ser Arg Gly Thr    #             40    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 10 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    -      Thr Arg Gln Met Ile Leu Ala Val - # Gly Gln    #   10    __________________________________________________________________________

What is claimed is:
 1. A process for the production of a D-α-amino acid,comprising:converting an N-carbamyl-D-α-amino acid of the formula:##STR7## wherein R is phenyl, hydroxy-substituted phenyl, substituted orunsubstituted alkyl or thienyl, to the corresponding D-α-amino acid ofthe formula: ##STR8## wherein R is as defined above in an aqueous mediumof a pH of 6 to 11, at a temperature of 20 to 85° C. in the presence ofa microbial decarbamylase in the form of a culture broth, living cells,dried cells, a homogenate, an extract or a crude enzyme preparation, andrecovering the resulting D-α-amino acid, wherein said microbialdecarbamylase is produced by a microorganism selected from the groupconsisting of: Comamonas sp. E 222 c, Alcaligenes sp. E 215,Sporosarcina sp. NCA 28-b, Blastobacter sp. A 17 p-4, Rhizobium sp. KNK1415, Rhizobium loti IFO 14779, Rhizobium meliloti IFO 14782, Rhizobiumfredii IFO 14780, Rhizobium galegae IFO 14965, Rhizobium huakuii IFO15243, Bradyrhyzobium japonicum IFO 14783, and Bradyrhyzobium sp. IFO15003.
 2. The process claimed in claim 1, wherein said microbialdecarbamylase is produced in a culture medium containing an additive forimproving decarbamylase production.
 3. The process claimed in claim 2,wherein the additive is selected from the group consisting of D-aminoacids, N-carbamyl-α-amino acids, 5-substituted hydantoins, pyrimidinemetabolites and metal ions.
 4. The process claimed in claim 2, whereinthe microorganism is Comamonas sp. E 222 C, and the additive is urea,β-ureidopropionic acid, D-phenylglycine or N-carbamyl-DL-methionine. 5.The process claimed in claim 2, wherein the microorganism isBlastobacter sp. A 17 p-4, and the additive is uracil, dihydrouracil,β-ureidopropionic acid, Li⁺, Cs⁺, Be⁺, Mg²⁺, Mn²⁺, Fe²⁺, Fe³⁺, Co²⁺ orAl³⁺.
 6. The process claimed in claim 1, wherein the aqueous medium isat pH 7.5 to 9.5.
 7. The process claimed in claim 1, wherein saidtemperature is at 40 to 50° C.
 8. The process claimed in claim 1,wherein the microorganism is Comamonas sp. E 222 C, Blastobacter sp. A17 p-4, Alcaligenes sp. E 215, Sporosarcina sp. NCA 28-b, Rhizobium sp.KNK 1415, Bradyrhizobium japonicum IFO 14783, Bradyrhizobium sp. IFO15003, Rhizobium loti IFO 14779, Rhizobium meliloti IFO 14782, Rhizobiumfredii IFO 14780, Rhizobium galegae IFO 14965, or Rhizobium huakuii IFO15243.
 9. A process for the production of a D-α-amino acidcomprising:converting an N-carbamyl-D-α-amino acid of the formula:##STR9## wherein R is phenyl, hydroxy-substituted phenyl, substituted orunsubstituted alkyl or thienyl, to the corresponding D-α-amino acid ofthe formula: ##STR10## wherein R is the same as defined above in anaqueous medium of pH 6 to 11, at a temperature of 10 to 60° C. in thepresence of a purified decarbamylase produced by Comamonas sp. E 222 cwhich is free or immobilized, and recovering the resulting D-α-aminoacid.
 10. The process as claimed in claim 9, wherein pH of said aqueousmedium is from 7.5 to 8.5 and said temperature is from 30° C. to 40° C.11. A process for the production of a D-α-amino acidcomprising:converting an N-carbamyl-D-α-amino acid of the formula:##STR11## wherein R is phenyl, hydroxy-substituted phenyl, substitutedor unsubstituted alkyl or thienyl, to the corresponding D-α-amino acidof the formula: ##STR12## wherein R is the same as defined above in anaqueous medium of pH 5 to 11, at a temperature of 20 to 80° C. in thepresence of a purified decarbamylase produced by Blastobacter sp. A 17p-4 which is free or immobilized, and recovering the resulting D-α-aminoacid.
 12. The process as claimed in claim 11, wherein pH of said aqueousmedium is from 6 to 10 and said temperature is from 45° C. to 55° C. 13.A process for the production of a D-α-amino acid, comprising:convertingan N-carbamyl-D-α-amino acid of the formula: ##STR13## wherein R isphenyl, hydroxy-substituted phenyl, substituted or unsubstituted alkylor thienyl, to the corresponding D-α-amino acid of the formula:##STR14## wherein R is as defined above in an aqueous medium of a pH of6 to 11, at a temperature of 20 to 85° C. in the presence of a microbialdecarbamylase in the form of a culture broth, living cells, dried cells,a homogenate, an extract or a crude enzyme preparation, and recoveringthe resulting D-α-amino acid, wherein said microbial decarbamylase isproduced by a microorganism selected from the group consisting of:Rhizobium loti, Rhizobium meliloti, Rhizobium fredii, Rhizobium galegae,Rhizobium huakuii, and Bradyrhizobium japonicum.